目的 拟利用藤石松Lycopodiastrum casuarinoides生药与饮片中微小RNA（miRNA）表达谱，鉴定其中热稳定存在的miRNA，为今后藤石松miRNA生物药效研究奠定基础。方法 选取藤石松的2种加工形态生药与饮片全株样品（包含茎、叶、分枝），采用Illumina HiSeq^TM 2500高通量测序方法，根据miRNA家族在不同物种中的保守性，将miRBase数据库中的已知植物miRNA与通过高通量测序获得的藤石松miRNA序列进行比对，利用生物信息学方法，注释藤石松中的所有miRNA序列，并鉴定生药与饮片之间的差异表达miRNA分子，同时完成它们相应的人类靶基因预测，此外，对这些miRNA及靶基因进行GO和KEGG功能注释分析。结果 生药组与饮片组分别获得9 898 332、10 099 918条序列，其长度基本呈正态分布，而且均在21、24 nt的位置上形成2个峰值。两组显著差异表达的miRNA共计25个，其中22个表达上调，3个表达下调。GO分析结果表明miRNA靶基因涉及富集与结合、催化、分子转导等分子功能以及细胞调控、代谢过程等生物过程。KEGG通路富集分析表明靶基因多集中于癌症与免疫相关通路，如pathways in cancer、proteoglycans in cancer等信号通路。结论 首次揭示了藤石松生药与饮片的miRNA表达谱，存在潜在的可利用的植物来源的稳定miRNA分子，为进一步研究藤石松中与癌症或免疫相关的miRNAs分子药理功能奠定基础。

Objective To detect the differential expression profile of miRNAs (microRNAs) in crude drugs and processed products of Lycopodiastrum casuarinoides and identify potential bioactive herbal-derived miRNAs. Methods The samples of the whole aerial tissues (including stems, leaves, branches) were collected in Yichun area of Jiangxi Province and were authenticated by relevant experts. General RNA was extracted from the crude drugs and processed products of L. casuarinoides, respectively. High-qualified small RNAs (sRNA) were isolated to be constructed the sRNA sequencing library. Then, the single-ended sequencing was conducted by using Illumina HiSeq^TM 2500 sequencing method. MiRNA characteristic of L. casuarinoides was analyzed by relevant bioinformatics. Results 9 898 332 and 10 099 918 clean reads were obtained from crude drugs group and processed products group, respectively. A total of 25 microRNAs were differentially expressed between crude drugs group and processed products groups with statistical significance, among which 22 were up regulated and three were down-regulated in processed products group compared to those in the crude drugs group. GO (gene ontology) analysis showed that their homo sapiens targets were enriched in catalytic binding, molecular transducer and KEGG (Kyoto encyclopedia of genes and genomes) analysis was shown for the target genes enriched in cancer and immunity-related pathways, such as pathways in cancer, proteoglycans in cancer. Conclusion The differential expression profile of microRNAs is first revealed by two processing forms of L. casuarinoides. This study suggests that the crude drugs and processed products miRNAs identified in this study will lay a foundation for exploring the pharmaceutical function of miRNAs in L. casuarinoides.

江西省对外科技合作重大项目（20151BDH80020）；江西省博士后基金课题（JX2016RC40）